Ribonucleases play important roles in various pathways of nucleic acid metabolism, including control of gene expression, mRNA surveillance and degradation and host defense mechanism against RNA viruses (1-3). Since the first ribonuclease was discovered as a heat stable enzyme from pancreas capable of digesting yeast RNA, a diverse panel of RNases has been characterized. However, unlike DNA restriction enzymes, a protein enzyme that cleaves RNA in a sequence-specific manner has not been found in nature. The known RNA endonucleases either specifically cleave their target through recognition of certain structures (e.g., RNase III family, RNase H or most ribozymes) (4-6), or have essentially no sequence specificity (e.g., RNase A cleaves after pyrimidine residues and RNase T1 cleaves after G residues) (7). The sequence specific cleavage of RNA can be achieved by a large multi-component complex such as the spliceosome or the RISC complex in RNAi pathway, each of which require guide RNA to recognize their targets (8, 9) and involve large protein/RNA assemblies, limiting their application in probing structured RNA or manipulating recombinant RNA in vitro.
Sequence specific cleavage of RNA has been achieved using engineered hammerhead ribozymes or RNA-cleaving DNAzymes (10). Both types of enzyme recognize their substrates through Watson-Crick binding arms of 6-12 nt, and therefore can achieve high target selectivity. However, these nucleic acid enzymes generally have low turnover rate (with kcat around 1 min−1) compared to protein enzymes, possibly due to tight binding to their substrates. In addition, the in vitro application of such nucleic acid enzymes is compromised by the high production cost and low stability of RNA, as well as the difficulty in controlling the folding of single stranded RNA or DNA.
The present invention overcomes previous shortcoming in the art by providing site specific RNA endonucleases and methods of their use.